Analysis of characteristics related to interstitial lung disease or pulmonary hypertension in patients with dermatomyositis

Abstract Introduction Dermatomyositis (DM) is often associated with interstitial lung disease (ILD) or pulmonary hypertension (PH). The aim of this study was to investigate the clinical characteristics of DM patients with ILD or PH. Methods This study retrospectively analysed the clinical characteristics of 372 patients with DM, including cytokines, lymphocyte subsets, immunoglobulin and complement. The DM patients were divided into different groups according to whether complicated with ILD, PH or anti‐melanoma differentiation‐associated gene 5 antibodies (MDA5). A qualitative and quantitative data analysis was performed. Results IgG, IgA and IgM in the DM‐associated ILD (ILD‐DM) were higher than that of the DM non‐complicating ILD (Non‐ILD‐DM) (p = 0.022, 0.002 and 0.029, respectively). Meanwhile, IL‐6 (p = 0.008) and IL‐10 (p = 0.001) were increased in the DM‐associated PH (PH‐DM) than in the DM non‐complicating PH (Non‐PH‐DM), while IL‐17 (p = 0.004), double positive (DP) cell ratio and B lymphocyte ratio were reduced in the PH‐DM. Moreover, the incidence of ILD and levels of C4 were higher in the DM with MDA5 (MDA5+ DM) than that of the DM without MDA5. Conclusion ILD‐DM has higher IgG, IgA and IgM than that of Non‐ILD‐DM. PH‐DM has higher IL‐6, IL‐10 and lower IL‐17, DP cell ratio and B lymphocyte ratio than that of Non‐PH‐DM.

Dermatomyositis (DM) is an idiopathic inflammatory disease that is frequently accompanied by inflammation, immune-mediated organ damage. 1 DM causes muscle, skin, joints, heart and lung damage, 1 among which lung injury predominantly presents interstitial lung disease (ILD) and pulmonary hypertension (PH). 2 ILD is a group of common heterogeneous diseases characterized by fibrosis or inflammation in the interstitial space. 3,4As the most serious complication of DM, ILD is mainly caused by impaired the exchange of gas leading to respiratory distress, which significantly increases the mortality rate of DM. 4,5 PH is a pulmonary vascular disease characterized by a variety of aetiologies, 3 which are linked to excessive vasoconstriction, vascular remodelling, inflammation and thrombosis in situ. 6Similarly, PH is closely related to DM complications and impairs DM prognosis. 7owever, few researches on which DM are more likely to develop PH have been found so far.Increasing researches suggest that immune cells and inflammatory cytokines play a crucial role in PH and ILD. 4,8Accordingly, this study retrospectively analysed the clinical characteristics of 372 patients with DM and compared the inflammatory, immunization and demographic characteristics of DM with ILD or PH.

| Patients
Patients with DM admitted to the First Affiliated Hospital of Kunming Medical University between August 2015 and September 2022 were included.DM was diagnosed according to the criteria proposed by Bohan and Peter in 1975. 9The following indicators for analysis were included, demographic characteristics (age, gender, ethnicity, smoking, drinking and family history), clinical data (diagnosis), relevant laboratory tests (cytokines, lymphocyte subsets, immunoglobulins and complement) and imaging manifestations (echocardiography and pulmonary CT).

| Comparison of patients' characteristics in different situations
The subjects were grouped according to different characteristics.According to Chinese ethnicity, all patients were divided into two groups, the Han population patients with DM and the ethnic minority patients with DM.According to the presence or absence of ILD, all patients were divided into two groups, the DM-associated ILD (ILD-DM) and the DM non-complicating ILD (Non-ILD-DM).ILD was diagnosed on the basis of clinical and physical examination results and imaging features. 1ccording to the presence or absence of PH, all patients were divided into two groups, the DM-associated PH (PH-DM) and the DM non-complicating PH (Non-PH-DM).PH was defined as a pulmonary artery systolic pressure (PASP) ≥ 30 mmHg at rest, measured by Doppler echocardiography. 10In addition, we conducted subgroup analyses of DM patients with or without ILD.According to the presence or absence of PH, all the ILD-DM patients were divided into two groups (PH/Non-PH) and all the Non-ILD-DM patients were divided into two groups (PH/Non-PH).Finally, according to the presence or absence of anti-melanoma differentiation-associated gene 5 antibodies (MDA5), all patients were divided into two groups, the DM with MDA5 (MDA5 + DM) and the DM without MDA5 (MDA5 À DM).MDA5 + was determined using an anti-MDA5 antibody detection method according to a linear immunoassay (Euroimmun [Germany] or

| Statistical analysis
Analysis was performed using SPSS 26.0.The chi-square test was used to compare the categorical variables.As for the continuous variables, the Shapiro-Wilk test was first performed to determine whether the data were normally distributed.If the data were normally distributed, the independent samples t-test was employed to compare it, and the data were presented as mean ± standard deviation (SD).If the data were not normally distributed, the Mann-Whitney U test was employed, and the data were presented as the median (25th percentile, 75th percentile).

| Baseline characteristics
A total of 372 patients with DM was included in this study.The demographic characteristics were listed in Table 1.

| DM patients in different ethnic groups in China
The Han population was significantly older than that of the ethnic minority DM patients (p < 0.001).In

| Comparison of the ILD-DM and non-ILD-DM characteristics
The ILD-DM was significantly older than the Non-ILD-DM (p = 0.019).There were no significant differences in cytokines between these two groups.Additionally, no significant differences in lymphocyte subsets were found.While IgG, IgA and IgM levels were considerably increased in the ILD-DM than in the Non-ILD-DM (p = 0.022, 0.002 and 0.029, respectively) (Table 3).

| Comparison of the PH-DM and non-PH-DM characteristics
The PH-DM was significantly older than Non-PH-DM (p < 0.001  4).
It is confirmed that PH is a common complication of ILD. 3 Analyses in subgroup were performed to exclude the effect of ILD on PH (Table 5).In the ILD-DM subgroup analysis, the Han population had a higher proportion of PH than that of Non-PH (p = 0.024).In addition, IL-6 and IL-10 levels were considerably higher than in Non-PH ( p = 0.004 and 0.039, respectively).Moreover, helper T (Th) cell ratio, Th cell counts, DP cell ratio, B lymphocyte ratio and B lymphocyte counts were lower in PH than in Non-PH ( p = 0.027, 0.044, 0.031, 0.045 and 0.029, respectively).In contrast, the NK cell ratio in PH was significantly increased than in Non-PH.Besides, the C4 was lower in PH than in Non-PH (0.20 ± 0.08 g/L vs. 0.24 ± 0.09 g/L p = 0.041).In the Non-ILD-DM subgroup analysis, PH was significantly older than Non-PH (p = 0.001).More importantly, IL-17 and interferon (IFN) -γ were significantly lower in PH than in Non-PH (p = 0.026 and 0.043, respectively).Also, the PCT was lower in PH than in Non-PH ( p = 0.031).However, lymphocyte subsets in this subgroup were no differences.Additionally, IgG levels were considerably elevated in PH (median [IQR]; 13.35 [10.83-16.28]g/L) than in Non-PH (median [IQR]; 11.70 [9.12-13.70]g/L (p = 0.016).

| Comparison of the MDA5 + DM and MDA5 À DM characteristics
The proportion of males with the MDA5 + DM was higher than that with the MDA5 À DM ( p = 0.043).Also, the MDA5 + DM owned more smoking history than the MDA5 À DM (p = 0.003).The MDA5 + DM had a higher rate of combining ILD than the MDA5 À DM (p < 0.001).Moreover, NK cell ratio and NK cell counts were significantly lower than in the MDA5 À DM (p = 0.004 and 0.002, respectively).Furthermore, IgA and C4 levels were considerably higher than in the MDA5 À DM (p = 0.014 and 0.005, respectively) (Table 6).

| DISCUSSION
The findings of this study revealed that the age of the Han population with DM patients was older than that of the ethnic minority, while PLT, PCT, IL-8 and IgM were lower in the Han population.In addition, IgG, IgA and IgM levels in the ILD-DM were higher than in the Non-ILD-DM.Moreover, IL-6 and IL-10 were increased in the PH-DM than in the Non-PH-DM, while PCT, IL-17, DP cell ratio and B lymphocyte ratio were reduced in the PH-DM.Meanwhile, the MDA5 + DM had more smoking history than the MDA5 À DM and the incidence of ILD was higher in the MDA5 + DM.Also, the MDA5 + DM had higher levels of IgA and C4, while the MDA5 + DM had lower levels of NK cell ratio and NK cell counts.
The result in the study showed that DM patients of the Han population were older than that of the ethnic minority, indicating that the age of clinical onset of DM in the ethnic minority tended to be younger.In addition, IL-8 and IgM were lower in patients with DM in the Han population than in the minority population.It is well known that IL-8 is a chemokine that plays an important role in most inflammatory conditions, 12 while IgM suppresses excessive inflammatory responses mediated by autoimmune mechanisms. 13However, what exactly are the reasons for the differences between IL-8 and IgM among ethnic groups need more research in the future.Meanwhile, PLT and PCT were lower than that of the ethnic minority in this study, which suggested that it was more important to pay attention to the changes of PLT and PCT in the Han population DM patients in clinical practice.Moreover, ethnicity-related T lymphocyte ILD, a common complication of DM, has a significant impact on prognosis. 1It is shown that advanced age is an independent risk factor for ILD in DM patients. 14Our study also showed that the age of the ILD-DM was higher than that of the Non-ILD-DM.
It is reported that older age group is more likely suffer from PH, which means that there is a correlation between PH and age. 15Our study showed that PH-DM patients were significantly older than Non-PH-DM patients.However, whether the correlation between PH and age is interfered with by DM is unclear and more researches are needed.It has been reported that IL-6 acts as both a proinflammatory and an anti-inflammatory cytokine and was associated with excessive cell proliferation and pulmonary vascular remodelling. 8,16Moreover, elevated IL-6 levels have been demonstrated in DM and in PH, where it played a major role in the inflammatory process of DM and in the pathogenesis of PH. 16,17 The same results were obtained in this study.IL-10 is one of the most important anti-inflammatory cytokines that inhibit excessive inflammatory processes. 17Elevated levels of IL-10 were found in patients with PH, which might be a counter-regulatory mechanism of lung tissue inflammation. 17Therefore, the elevated IL-10 level in PH-DM patients in our study might also be related to the anti-regulatory mechanism of lung tissue inflammation.It has been presented that IL-17 was highly expressed in both DM and PH and this elevation was also associated with the pathogenesis of DM. 18,19 Contrary to the findings of other researches, our study found that IL-17 was significantly lower in PH than in Non-PH, both in the overall analysis and in the subgroup analysis.We speculated that such opposite findings might be due to differences in the characteristics or ethnicity of the study populations.It has been reported that PLT activation played an important role in the pathogenesis of PH, and PCT was a marker of PLT activation. 6,20Our study found that PH had lower PCT than Non-PH, which indicated that PCT might be linked to the aetiology of PH in DM patients.Also, some studies suggested that T and B lymphocytes might be intimately associated with the development of PH. 21In this study, DP cells and B lymphocytes were shown to be considerably lower in PH than in Non-PH, and differences in these indicators were also seen in the subgroup analysis of the ILD-DM.Besides that, it has been reported that peripheral blood T lymphocytopenia was a prevalent clinical phenomenon in DM and its deficiency also increased the tendency to develop PH in animal experiments. 22,23Therefore, we speculated that PH might be caused by T lymphocytopenia in DM.Meanwhile, it has been suggested that the decrease in B lymphocytes might be due to the accumulation of B lymphocytes in inflamed muscle tissue in patients with DM, which caused B lymphocytes to decrease in the peripheral blood. 24Since B lymphocytes were also implicated in the pathogenesis of PH, we hypothesized that the incidence of PH might also be connected to the decline in peripheral B lymphocytes in DM patients.Previous reports have shown that NK cells were reduced in PH alone. 22However, our study showed that NK cells were considerably higher in PH than in Non-PH, suggesting that a possible difference in the pathogenesis between PH caused by DM and PH alone.Finally, our study showed that IgA levels were significantly higher in the PH-DM than in the Non-PH-DM, which seemed to suggest that IgA was involved in the aetiology of PH-DM.However, we performed subgroup analysis and found that there was no difference in IgA levels between PH and Non-PH in both the ILD-DM and the Non-ILD-DM subgroups.Therefore, we made the assumption that IgA might not be a major factor causing PH in DM patients.Currently, there are contradictory perspectives on whether there is an association between MDA5 and gender in patients with DM.Research in China reported no correlation between the incidence of anti-MDA5 antibodies and the patients' gender, 25 while a study in Europe concluded that patients with MDA5 + DM were mainly female. 11Our study showed that the proportion of male with the MDA5 + DM was higher than that with the MDA5 À DM.It seemed that race might have an influence on the connection between MDA5 and gender.In addition, there are no research studies that demonstrate a correlation between smoking and MDA5 + DM.Our study showed that the MDA5 + DM had more smoking history than the MDA5 À DM.Nevertheless, due to the small number of smokers we included, further research is needed to explain whether smoking is related to MDA5 + DM.A growing number of studies have shown that one of the typical features of MDA5 + DM was the presence of ILD. 26 Anti-MDA5 antibodies might also help with the diagnosis of ILD when found in DM. 26 Moreover, it has been reported that peripheral lymphopenia was prevalent and easily detected in MDA5 + DM-ILD. 27Our study also found that NK cells were significantly lower in the MDA5 + DM and the changes might be due to the transfer of lymphocytes to the lungs to participate in the local immune response. 27Also, it has been shown that anti-MDA5 antibodies in serum were mainly present in the form of IgA and other forms. 28Because IgA levels in our research were considerably higher in the MDA5 + DM than in the MDA5 À DM, we hypothesized that anti-MDA5 antibodies in MDA5 + DM might be present in the form of IgA.In this study, C4 levels were significantly higher in the MDA5 + DM than in the MDA5 À DM, suggesting that elevated C4 levels might be associated with anti-MDA5 antibodies in DM, which was consistent with other findings. 29his study has some limitations.First, this study was a small, retrospective study with limited case data collected.Also, being a non-multicentre study, only DM cases from one hospital were included, and the possibility of unintentional selection bias could not be completely excluded.Therefore, prospective studies with large sample sizes are needed to confirm our conclusions in future.Secondly, data related to the prognosis of patients with DM were not collected in this study and the findings were not compared with clinical long-term prognosis.Thus, not allowing further assessment of whether the influencing factors included had an impact on disease progression and outcome in patients with DM.In addition, the age difference between the Han population and the Minority population may be a confounding factor for the study, so the differences of other parameters such as Tc/Ts cell counts between the two populations may also be due to age.Accordingly, future research is needed on the relationship between ethnicity and other factors and to exclude the confounding factor of age.Indeed, differences in immunoglobulins between the two groups may be influenced by Sjogren syndrome (SS).However, because some patients in our study were not tested for SS-specific antibodies, the diagnosis of SS was failed to be excluded from the collection of medical records.Accordingly, the subsequent research needs to exclude the effect of SS on DM.Finally, no correlation analysis was performed in this study, and it was not possible to grasp the correlation and closeness between the clinical features

| CONCLUSION
Han population is older than ethnic minority in China for DM patients and has lower levels of IL-8 and IgM than ethnic minority.Also, ILD-DM has higher levels of IgG, IgM and IgA than that of Non-ILD-DM.Additionally, PH-DM has higher levels of IL-6, IL-10 and lower levels of IL-17 than that of Non-PH-DM.Meanwhile, it is possible that the decrease in DP cell ratio and B lymphocyte ratio are associated with the development of PH in DM.Moreover, MDA5 + DM is more likely to have combined ILD.Anti-MDA5 antibodies in MDA5 + DM may be present in the form of IgA and high levels of C4 may be associated with anti-MDA5 antibodies.

AUTHOR CONTRIBUTIONS
Chulin Wang designed and performed the research, collected and analysed the data, and wrote the paper.Han Yang collected and analysed the data and wrote the paper.Tongfen Li designed the research and wrote the paper.Yiqiong Wen designed the research and collected the data.Heran Yang collected and analysed the data.Weifeng Tang performed the research and analysed the data.Lin Li designed and performed research.Shibo Sun designed and performed the research, analysed the data, and wrote the paper.T A B L E 6 (Continued) MDA5 + (n = 30) MDA5 À (n = 342) double negative (CD4-CD8-) T-cells; DP cell, double positive (CD4 + CD8+) T-cell; IFN, interferon; ILD, interstitial lung disease; MDA5, antimelanoma differentiation-associated gene 5 antibodies; MPV, mean platelet volume; NK cell, natural killer cell; PCT, plateletcrit; PDW, platelet volume distribution width; PE, pulmonary embolism; PH, pulmonary hypertension; P-LCR, platelet-large cell ratio; PLT, platelet; Tc/Ts, cytotoxic/suppressor T-cell; Th cell, helper T-cell; TNF, tumour necrosis factor.*p < 0.05.# p < 0.001.
Comparison of the ILD-DM and non-ILD-DM characteristics.
T A B L E 4 Comparison of the ILD-DM subgroup and non-ILD-DM subgroup characteristics.
T A B L E 5 Comparison of the MDA5 + DM and MDA5 À DM characteristics.